The present invention relates to a novel peptide useful as a therapeutic agent of diseases such as tumor and arteriosclerosis, for which cellular abnormal growth is responsible, by inhibiting the activity of a transcription factor E2F which regulates the transcription of gene groups involved in the progress of cell cycle to thereby suppress cell growth.
E2F is a transcription factor of importance for the transcription of a great number of genes involved in the progress of cell cycle and serves as a target protein of tumor suppression gene product Rb [EMBO J., 9, 2179 (1990); Cell, 65, 1053 (1991)]. As proteins comprising E2F, E2F family and DP family have been known. Up to now, five molecules of the E2F family, namely E2F 1 to 5 have been identified, while two molecules of the DP family, namely DP1 and 2, have also been identified. It has been believed that the out of control expression or activity of E2F is deeply involved in the carcinogenesis of a great number of cells [Science, 258, 424 (1992); Trends in Biological Science, 19, 108 (1994)]. It has also been reported that the inhibition of the transcriptional activity of E2F can suppress the growth of smooth muscle cells, which works as the cause of arteriosclerosis [Proc. Natl. Acad. Sci. USA., 92, 5855 (1995)]. Thus, the substance suppressing the E2F activity is useful as a therapeutic agent of tumor or diseases involving the abnormal growth of smooth muscle cells or the like, such as arteriosclerosis. Additionally, the substance may also be effective widely for autoimmune diseases which are exacerbated due to the growth of synovial cell, such as chronic rheumatoid arthritis, or diseases occurring because of the abnormal growth of mesangium cell, such as nephropathy. As to E2F suppressing agents, nucleic acid based compounds have been known, including antisense RNA [Cancer Res., 54, 1402 (1994)] and decoy based on the E2F binding sequence DNA [Proc. Natl. Acad. Sci. USA., 92, 5855 (1995)]. However, no peptides have been known yet as such suppressing agents.
In accordance with the present invention, it is provided a compound represented by the general formula (I);
R1xe2x80x94Axe2x80x94R2xe2x80x83xe2x80x83(I)
(wherein R1 represents substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or , unsubstituted heteroaryloxycarbonyl or a hydrogen atom; R2 represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino; and A represents a peptide sequence comprising a partial amino acid sequence having at least 12 continuous residues in the sequence of the dimerization region or DNA binding region of each E2F family) [simply referred to as xe2x80x9cCompound Ixe2x80x9d hereinafter] or a pharmaceutically acceptable salt thereof. In accordance with the present invention, a pharmaceutical composition comprising the Compound (I) or a pharmaceutically acceptable salt thereof is provided.
In the definition of each group in the formula (I), the alkanoyl includes alkanoyl groups with 1 to 20 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, lauroyl, and icosanoyl.
The substituted alkanoyl has, the same or different, 1 to 3 substituents such as hydroxy, carboxyl, alicyclic alkyl, substituted or unsubstituted phenyl or fluorenyl.
Herein, the alicylic alkyl includes alicyclic alkyl groups with 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
The substituted phenyl has, the same or different, 1 to 3 substituents such as alkyl, alkoxy, hydroxy, nitro, sulfo, cyano or halogen. The alkyl and the alkyl moiety of the alkoxy include alkyl groups with 1 to 20 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, decyl, dodecyl, and icosyl; and the halogen includes each atom of fluorine, chlorine, bromine and iodine.
The aryl moiety of the aroyl and the aryloxycarbonyl includes e.g. phenyl and naphthyl. Each of the substituted aroyl and the substituted aryloxycarbonyl has, the same or different, 1 to 3 substituents such as alkyl, alkoxy, hydroxy, nitro, sulfo, or halogen. The alkyl and the alkyl moiety of the alkoxy and the halogen have the same meanings as defined above, respectively.
The heteroaryl moiety of the heteroarylcarbonyl and the heteroaryloxycarbonyl includes e.g., furyl, thienyl, pyridyl, pyrrolyl, pyrazolyl, pyrimidinyl, pyradinyl, indolyl, quinolyl, isoquinolyl, and quinazolyl. Each of the substituted heteroarylcarbonyl and the substituted heteroaryloxycarbonyl has the same substituents as defined for the substituents of the substituted aroyl.
The alkyl moiety of the alkoxycarbonyl and the alkoxy means the same as defined above. The substituents of the substituted alkoxycarbonyl and the substituted alkoxy include e.g. hydroxy, carboxy, carbamoyl, alicyclic alkyl, substituted or unsubstituted phenyl and fluorenyl. The alicyclic alkyl and the substituent of the substituted phenyl mean the same as defined above, respectively.
The substituted amino has, the same or different, 1 to 2 substituents such as substituted or unsubstituted alkyl, or substituted or unsubstituted aryl. The alkyl and the aryl have individually the same meanings as defined above. The substituents of the substituted alkyl include e.g. hydroxy, carboxy, carbamoyl, alicyclic alkyl and a phenyl group. The alicyclic alkyl has the same meaning as defined above. The substituted aryl has, the same or different, 1 to 3 substituents such as alkyl, alkoxy, hydroxy, nitro, sulfo or halogen. The alkyl and the alkyl moiety of the alkoxy, and the halogen have the same meanings as defined above, respectively.
In accordance with the present invention, the term xe2x80x9cE2Fxe2x80x9d means a protein which binds to the E2F binding sequence in DNA and influences the promoter activity around the sequence. E2F is composed of the E2F family and DP family, and each family has a dimerization region and a DNA binding region. The sequences of the dimerization region and DNA binding region in each family of E2F are described in Cell, 70, 337 (1992); Cell, 70, 351 (1992); Mol. Cell. Biol., 13, 7802 (1993); Mol. Cell. Biol., 13, 7813 (1993); Genes and Dev., 8, 2680 (1994); Genes and Dev., 8, 2665 (1994); Proc. Natl. Acad. Sci. USA., 92, 2403 (1995); Nature, 362, 83 (1993); Mol. Cell. Biol., 15, 2536 (1995) and the like.
The peptide sequence comprising a partial amino acid sequence having at least 12 continuous residues in the sequence of the dimerization region of the E2F includes SEQ ID NO:25, e.g., a sequence represented by the general formula (Ia);
(wherein xe2x80x9cn""s in individual amino acid residues are the same or different, and represent 0 or 1; X1, X8, X27 and X28 are the same or different, representing Leu or Ile; X2 represents Asn or Lys; X3 represents Trp, Lys, Leu, Ala or Glu; X5 represents Ala or Ser; X6 represents Glu, Asp or Asn; X7 represents Val, Thr or Arg; X9 represents Lys, Asp, Ala or His; X26 represents Gln, His, Gly, Asp or Asn; and X29 represents Ala, Arg, Lys or Glu), and SEQ ID NO: 26, e.g., a sequence represented by the general formula (Ib);
(wherein xe2x80x9cm""sxe2x80x9d in individual amino acid residues are the same or different, and represent 0 or 1; Y1 represents Asn, Thr, Ala or Tyr; Y2 represents Glu or Asp; Y3 represents Ser or Asn; Y5 represents Ala or Asn; Y6 represents Tyr or Cys; Y9 represents Lys or Glu; Y25 represents Met or Ile; and Y27 represents Ile or Val.)
The peptide sequence comprising a partial amino acid sequence having at least 12 continuous residues in the sequence of the DNA binding region of the E2F includes SEQ ID NO: 27, e.g. a sequence represented by the general formula (Ic);
(wherein xe2x80x9cp""sxe2x80x9d in individual amino acid residues are the same or different, and represent 0 or 1; Z1 represents Ala, Phe or Pro; Z2 represents Arg, Lys or Gln; Z3, Z15 and Z21 are the same or different, representing Gly or Pro; Z4 represents Arg, Lys, Met or Pro; Z5 represents Gly, Cys, Ala or Gln; Z6 represents Ala, Arg or Glu; Z7 represents Ala, Ile or Gln; Z8 represents Ala, Gly or Arg; Z9 represents Leu, Val or Pro; Z10 represents Asp, Arg or Gln; Z11 represents Gly, Ser, Ala or Pro; Z12 represents Leu or Pro; Z13 represents Asp, His or Pro; Z14 represents Ser or Pro; Z16 represents Gln or Lys; Z17 represents Gly, Thr or Leu; Z18 represents Gly, Pro or Val; Z19 represents Gly or Lys; Z20 represents Ala or Ser; Z22 represents Gly or Ser; Z23 represents Gly, Glu or Thr; Z24 represents Arg, Lys, Ser or Pro; Z25 represents Ser or Thr; Z27 represents His or Tyr; Z28 represents Asp or Glu; Z29 and Z36 are the same or different, representing Lys or Thr; Z32 represents Gly or Asn; Z34 represents Leu or Thr; Z37 represents Arg or Lys; Z39 represents Ile, Leu or Val; and Z40 represents Glu, Gln, Ser or Tyr)
The pharmaceutically acceptable salt of the Compound (I) includes acid addition salts, metal salts, organic base addition salts and the like. The acid addition salts include inorganic salts such as hydrochloride, sulfate and phosphate; and organic salts such as acetate, maleate, fumarate, tartrate, and citrate. The metal salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; aluminium salt, zinc salt and the like. The organic base addition salts include salts of primary amines such as methylamine, ethylamine and aniline; secondary amines such as dimethylamine, diethylamine, pyrrolidine, piperidine, morpholine and piperazine; and tertiary amines such as trimethylamine, triethylamine, N,N-dimethylaniline and pyridine; and ammonium salts.
The present invention will now be described in. detail hereinbelow.
The abbreviations of amino acids and the protective groups thereof follow the recommendations by IUPAC-IUB Joint Commission on Biochemical Nomenclature [Eur. J. Biochem., 138, 9 (1984)].
The following abbreviations represent the following corresponding amino acids and protective groups thereof, unless otherwise stated.
Gly; Glycine
Ala; L-Alanine
Thr; L-Threonine
Asp; L-Aspartic acid
Asn; L-Asparagine
Asx; L-Aspartic acid or L-asparagine
Glu; L-Glutamic acid
Gln; L-Glutamine
Glx; L-Glutamic acid or L-glutamine
Trp; L-Tryptophan
Val; L-Valine
Leu; L-Leucine
Ser; L-Serine
Met; L-Methionine
Ile; L-Isoleucine
Phe; L-Phenylalanine
Tyr; L-Tyrosine
Lys; L-Lysine
Arg; L-Arginine
His; L-Histidine
Pro; L-Proline
Fmos; 9-Fluorenylmethyloxycarbonyl
t-Bu; t-Butyl
Trt; Trityl
Pmc; 2, 2, 5, 7, 8-Pentamethylchroman-6-sulfonyl
Boc; t-Butyloxycarbonyl
Ac; Acetyl.
The following abbreviations represent the corresponding side-chain-protected amino acids as follows.
Fmoc-Asp(Ot-Bu)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-L-aspartic acid xcex2-t-butylester
Fmoc-Glu(Ot-Bu)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-L-glutamic acid xcex3-t-butylester
Fmoc-Thr(Ot-Bu)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-threonine
Fmoc-Ser(t-Bu)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-serine
Fmoc-Tyr(t-Bu)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-tyrosine
Fmoc-Lys(Boc)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-Nxcex5-t-butyloxycarbonyl-L-lysine
Fmoc-Asn(Trt)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-Nxcex3-trityl-L-asparagine
Fmoc-Gln(Trt)-OH; Nxcex1-9 -Fluorenylmethyloxycarbonyl-Nxcex4-trityl-L-glutamine
Fmoc-Arg(Pmc)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-Ng-2,2, 5,7,8-pentamethylchroman-6-sulfonyl-L-arginine
Fmoc-His(Trt)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-Nim-trityl-L-glutamine
Fmoc-Trp(Boc)-OH; Nxcex1-9-Fluorenylmethyloxycarbonyl-Nind-t-butyloxycarbonyl-L-tryptophane
The following abbreviations represent the corresponding reaction solvents and reaction reagents and the like as follows.
PyBOP; Benzotriazol-1-yloxytrispyrrolidinophosphonium hexafluorophosphate
HOBt; 1-Hydroxybenzotriazole
NMM; N-Methylmorpholine
DMF; N,N-Dimethylformamide
TFA; Trifluoroacetic acid
HBTU; 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
DIEA; N,N-Diisopropylethylamine
NMP; N-Methylpyrrolidone.
The method for producing the Compound (I) will now be described below.
The Compound (I) can be synthesized by general liquid phase or solid phase peptide synthetic methods [Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen (1985)] or a combination thereof. Furthermore, an automatic peptide synthesizer may be used. The peptide synthesis on a commercially available peptide synthesizer, e.g. a peptide synthesizer manufactured by Shimadzu Corporation, a peptide synthesizer manufactured by Applied BioSystems Inc., USA (ABI Inc.), and a peptide synthesizer manufactured by Advanced ChemTech Inc., USA (ACT Inc.) can be done by using Nxcex1-9-fluorenylmethyloxycarbonyl amino acids or Nxcex1-t-butyloxycarbonyl amino acids with an appropriately protected side chain, according to the synthetic programs for the individual peptide synthesizers.
The protected amino acid as the starting materials of the Compound (I) and carrier resin can be available from ABI Inc., Shimadzu Corporation, Kokusan Chemical Works Co., Ltd., Nova Biochem Co., Watanabe Chemical Industries, Ltd., ACT Inc. or Peptide Institute Inc.
The Compound (I) thus obtained can be purified by high-performance liquid chromatography (referred to as HPLC hereinbelow) by using reverse-phase silica gel columns such as C-4, C-8 or C-18 type, column chromatography such as gel filtration with partition resin, adsorption resin, ion exchange resin, silica gel, chemically modified silica gel, reverse-phase silica gel, alumina, diamatoceous earth or magnesium silicate, or thin-layer chromatography.
The pharmaceutically acceptable salt of the Compound (I) can be obtained in a conventional manner. More specifically, the acid addition salt or organic base addition salt of the Compound (I) can be obtaiend by dissolving the Compound (I) in an aqueous solution of an acid or an organic base corresponding thereto and then freeze-drying the solution. The metal salt of the Compound (I) can be obtained by dissolving the Compound (I) in an aqueous solution containing the corresponding metal ion and purifying the solution by gel filtration or by HPLC.
Specific examples of the Compound (I) are shown in Table 1. Specific examples of the compound being represented by the general formula (I) having the amino acid sequence represented by the general formula (Ia) include compounds represented by Sequence ID Nos.1, 2 and 20, which are referred to as Compounds Ia-1, Ia-2 and Ia-3, respectively. Specific examples of the compound being represented by the general formula (I) having the amino acid sequence represented by the general formula (Ib) include compounds represented by Sequence ID Nos.3, 4 and 21, which are referred to as Compounds Ib-1, Ib-2 and Ib-3, respectively. Specific examples of the compound being represented by the general formula (I) having the amino acid sequence represented by the general formula (Ic) include a compound represented by SEQ ID No.5. which is referred to as Compound Ic-1.
In the table, Ac represents an acetyl group; and La represents a lauroyl group.